Fig. 6

Connectivity map analysis of AK4 gene signature identifies withaferin-A as a potent anti-metastatic agent for NSCLC. a Identification of structurally similar drug candidates with the best reverse AK4 gene expression enrichment score by querying the connectivity map. b Invasion assay of A549 and CL1-5 cells treated with the corresponding IC10 doses of drug candidates. The data are expressed as percent inhibition compared with DMSO as the vehicle control. **P ≤ 0.01 c WB analysis of HIF-1α and AK4 protein levels in A549, CL1-5, CL1-0 vector-, and AK4-expressing cells treated with or without withaferin-A under Nx or Hx. d Gross view (formalin-fixed) and H&E staining images of lungs from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of tumor weight in lungs of mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 28 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). e Gross view (formalin-fixed) and H&E staining images of livers from mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (top). Quantification of liver nodule number in mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 30 after orthotopic injection of CL1-0 cells overexpressing AK4 (bottom). f Diagram depicting a working model of AK4-induced HIF-1α stabilization via intracellular ROS elevation, leading to subsequent EMT and metastasis. Targeting of the AK4-HIF-1α axis by withaferin-A impairs lung cancer metastasis. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. *P ≤ 0.05; **P ≤ 0.01